Human cathepsins K and S are members of the protein family of cysteine cathepsins with very
similar tertiary structures. They have numerous physiological roles in human body, particularly
the most important biological role of cathepsin K is degradation of collagen fibre in bone tissue,
whereas cathepsin S plays a very important physiological role in regulating antigen presentation.
Cathepsins K and S have to be sufficiently regulated since their increased enzyme activities could
contribute to the progression of numerous diseases. Considering certain inhibitors of individual
cathepsins K and S have reached clinical trials, these two enzymes represent important potential
therapeutic targets for drug design. Targeting evolutionary less conserved allosteric sites could
enable more selective inhibition, which could cause less side effects and could therefore represent
an alternative approach for therapeutic purposes.
After detailed characterization of hyperbolic inhibitor of cathepsin K, methyl [(3RS)-2,5-
dioxopyrrolidin-3-yl]glycinate (3a), it was shown that it is selective against cathepsin K over
human cathepsins B, L, S in V. Since 3a displayed weak inhibitory effect for cathepsin S, as well,
we designed and prepared potential inhibitors of cathepsins K and S based on 3a, which would
bind with higher affinities and more selectively to individual enzyme. For the most potent
inhibitors, more accurate values of affinities and mechanisms of action were determined. The aim
of the research work was also to design and prepare mutant forms of enzymes based on
superposition of tertiary structures of human cathepsins K and S and based on potential allosteric
pathways of human cathepsin K, predicted by molecular dynamic simulations, as well, and to
identify residues important for their allosteric regulation.
We designed and prepared compounds with substituents at two sites for diversification of the
succinimide scaffold of compound 3a. Among them, methyl [(3R)-2,5-dioxopyrrolidin-3-yl]-L-threoninate (R-3c) was characterized as a hyperbolic inhibitor of cathepsin S and its absolute
(2S,3R,3'R)-configuration was determined by X-ray structural analysis. Then, we designed and
prepared cyclic derivatives of the compound R-3c and among four derivatives, (3’RS)-3-
{[(1S,2R)-2- hydroxycyclohexyl]amino}pyrrolidine-2,5-dione ((1S,2R)-7) was identified and
characterized as the most potent hyperbolic selective inhibitor of cathepsin S. It was shown by
enzyme kinetics that allosteric modifiers of cathepsin K or S, compounds 3a, R-3c and (1S,2R)-
7, act via similar mechanisms as allosteric effectors of cathepsin K NSC13345 in NSC94914.
Therefore, the first small-molecule allosteric effectors of cathepsin S (R-3c and (1S,2R)-7) were
identified and characterized. Apart from that, one part of the hypotheses of our research work was
confirmed since it was shown that the compound (1S,2R)-7 selectively inhibits cathepsin S over
cathepsin K. The mechanisms of action, which were determined for the compounds, are consistent
with the results of compound screening on degradation of macromolecular substrates via
cathepsins K and S. Hyperbolic inhibitors 3a, R-3c in (1S,2R)-7 have the potential to be used for
further development to lead compounds, which could be used for research purposes.
Mutant forms of cathepsins K and S with residues substituted by alanine residue in predicted
allosteric pathway and allosteric site of cathepsin K as well as its homologous site of cathepsin S
were designed and prepared. We have shown that residues in predicted allosteric pathway N202
and N208 are important for allosteric communication in cathepsin K between allosteric site and
site S.
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